Upon stimulation of platelets, a number of different events occur. One of these events is the activation of a calcium-activated neutral protease (CANP). This enzyme catalyzes the limited proteolysis of several proteins which are intimately involved in platelet function: glycoprotein Ib, actin-binding protein, von Willebrand factor, figrinogen and protein kinase C. In order to increase our knowledge of the mechanisms involved in platelet activation, it is essential that we understand the structure and function of these proteins and how they are modified in response to stimuli. The objectives of this proposal are to characterize CANP and to investigate mechanisms by which CANP may be involved in platelet activation. CANP has been purified from human platelets and partially characterized by other investigators. In this investigation, CANP will be purified and further characterized with respect to substrate specificity and the relationship between the two forms of CANP. One form of CANP (which requires a high concentration of calcium ions) may be converted by autoproteolysis to the other form (which requires a lower, more physiological, concentration of calcium ions). An important component of the methodology used in this project will be the development of a murine monoclonal antibody against CANP. The monoclonal antibody will be used to determine the subcellular localization of CANP by immunoelectron microscopy. This proposal will address several specific mechanisms by which CANP may be involved in platelet activation. Are the CANP-sensitive high molecular weight complexes of glycoprotein Ib and actin-binding protein degraded upon activation of platelets? Is the activation of protein kinase C by CANP involved in any of the various mechanisms of platelet activation by different stimuli? Does CANP play a role in the activation of phospholipase C? Is phospholipase A2 present in platelet membranes as an inactive proenzyme which can be activated by CANP? This investigation will lead to a better understanding of the events that take place upon platelet stimulation and aggregation and thus provide new insight into the etiology of thrombotic and hemostatic disorders.